This proposal exploits the unique advantages of the budding yeast Saccharomyces cerevisiae by combining a cellular, genetic and biochemical approach to examine the role of the formin Bni1 and Bnr1 in the generation of actin cables. The Pellman lab has recently developed a Bni1-3GFP constructs that leads to improved imaging of the behavior of Bni1 in vivo. Bni1-3GFP displays linear retrograde movement, which is dependent on actively polymerizing actin cables. In contrast Bnr1 appears to remain stationary at the bud neck. These fluorescent constructs, in conjunction with Abp140-3CFP, will be used to examine the differences in Bni1 cables and Bnr1 cables. The model of formin activity suggests that the rate of formin- induced nucleation controls the rate of cable extension. To examine this hypothesis, I will generate Bni1 FH2 domain mutants with impaired actin assembly and compare the rate of cable elongation (via Abp140- 3GFP) with wild type Bni1. Preliminary evidence from the Pellman lab demonstrates that Bud6 binds to microtubules (MT). I will further characterize this interaction and determine whether or not MT binding by Bud6 depends on the formin-mediated activity of Bud6. [unreadable] [unreadable] [unreadable]